Enhancing [177Lu]Lu-DOTA-TATE therapeutic efficacy in vitro by combining it with metronomic chemotherapeutics

Background Peptide receptor radionuclide therapy (PRRT) uses [177Lu]Lu-[DOTA0-Tyr3]octreotate ([177Lu]Lu-DOTA-TATE) to treat patients with neuroendocrine tumours (NETs) overexpressing the somatostatin receptor 2A (SSTR2A). It has shown significant short-term improvements in survival and symptom alleviation, but there remains room for improvement. Here, we investigated whether combining [177Lu]Lu-DOTA-TATE with chemotherapeutics enhanced the in vitro therapeutic efficacy of [177Lu]Lu-DOTA-TATE. Results Transfected human osteosarcoma (U2OS + SSTR2A, high SSTR2A expression) and pancreatic NET (BON1 + STTR2A, medium SSTR2A expression) cells were subjected to hydroxyurea, gemcitabine or triapine for 24 h at 37oC and 5% CO2. Cells were then recovered for 4 h prior to a 24-hour incubation with 0.7–1.03 MBq [177Lu]Lu-DOTA-TATE (25 nM) for uptake and metabolic viability studies. Incubation of U2OS + SSTR2A cells with hydroxyurea, gemcitabine, and triapine enhanced uptake of [177Lu]Lu-DOTA-TATE from 0.2 ± 0.1 in untreated cells to 0.4 ± 0.1, 1.1 ± 0.2, and 0.9 ± 0.2 Bq/cell in U2OS + SSTR2A cells, respectively. Cell viability post treatment with [177Lu]Lu-DOTA-TATE in cells pre-treated with chemotherapeutics was decreased compared to cells treated with [177Lu]Lu-DOTA-TATE monotherapy. For example, the viability of U2OS + SSTR2A cells incubated with [177Lu]Lu-DOTA-TATE decreased from 59.5 ± 22.3% to 18.8 ± 5.2% when pre-treated with hydroxyurea. Control conditions showed no reduced metabolic viability. Cells were also harvested to assess cell cycle progression, SSTR2A expression, and cell size by flow cytometry. Chemotherapeutics increased SSTR2A expression and cell size in U2OS + SSTR2A and BON1 + STTR2A cells. The S-phase sub-population of asynchronous U2OS + SSTR2A cell cultures was increased from 45.5 ± 3.3% to 84.8 ± 2.5%, 85.9 ± 1.9%, and 86.6 ± 2.2% when treated with hydroxyurea, gemcitabine, and triapine, respectively. Conclusions Hydroxyurea, gemcitabine and triapine all increased cell size, SSTR2A expression, and [177Lu]Lu-DOTA-TATE uptake, whilst reducing cell metabolic viability in U2OS + SSTR2A cells when compared to [177Lu]Lu-DOTA-TATE monotherapy. Further investigations could transform patient care and positively increase outcomes for patients treated with [177Lu]Lu-DOTA-TATE. Supplementary Information The online version contains supplementary material available at 10.1186/s13550-024-01135-0.


Introduction
Neuroendocrine tumours (NETs) are relatively rare and hard to treat.They originate in specialized neuroendocrine cells and can occur anywhere in the body.As 80-100% of gastric NETs overexpress SSTR2A [1], anticancer therapies targeting the SSTR2A receptor remain an excellent strategy.Amongst these, [ 177 Lu]Lu-[DOTA 0 -Tyr 3 ]octreotate ([ 177 Lu]Lu-DOTA-TATE), which binds and irradiates NETs with SSTR2A overexpression, is now a well-established therapeutic option for patients with advanced gastroenteropancreatic NETs.In the landmark Phase 3 clinical trial, NETTER-1, median overall survival was enhanced in NET patients receiving [ 177 Lu] Lu-DOTA-TATE from 36 to 48 months, however objective tumour responses were observed in only 18% of patients [2][3][4][5].Therefore, long-term outlook for most patients remains poor and there is still room for improvement.Work is ongoing to enhance the tumour uptake or therapeutic efficacy of [ 177 Lu]Lu-DOTA-TATE through, for example, photothermal therapy or combinations with DNA damage repair protein inhibitors or other radiosensitisers [6][7][8][9][10][11].
As the therapeutic efficacy of peptide receptor radionuclide therapy (PRRT) can still be enhanced, here we investigated an approach combining [ 177 Lu]Lu-DOTA-TATE with commonly used chemotherapeutics in NET patients.As well as somatostatin analogues and targeted therapies such as everolimus, and sunitinib, patients with NETs can also receive DNA-alkylating chemotherapeutics such as temozolomide and streptozotocin and those targeting DNA synthesis, such as gemcitabine and triapine, both of which act through inhibiting ribonucleotide reductase [12,13].Ribonucleotide reductase inhibitors decrease the synthesis of deoxyribonucleotides and thereby block DNA synthesis; they have often been investigated as a radiosensitisers [12].Gemcitabine has recently been tested in combination with oxaliplatin for gastroenteropancreatic-/pan-NETs [14], while triapine is currently under investigation in ongoing phase 1 (NCT04234568) and phase 2 (NCT05724108) clinical trials for treating NET patients in combination with [ 177 Lu] Lu-DOTA-TATE PRRT.
Here, we thus investigated whether treatment combinations with gemcitabine and triapine, particularly at metronomic levels, increased the therapeutic efficacy of [ 177 Lu]Lu-DOTA-TATE in SSTR2-expressing and nonexpressing cells.Metronomic chemotherapeutics here refers to the treatment in which low doses of anticancer drugs are given on a continuous or frequent, regular schedule.Alongside these drugs, another effective ribonucleotide reductase inhibitor, namely hydroxyurea [15][16][17][18][19], was also investigated for its potential to enhance the therapeutic efficacy of [ 177 Lu]Lu-DOTA-TATE using the same strategy.
SSTR2A expression in cells was confirmed microscopically and by flow cytometry (Figure S1; see supplementary methods for details [7,11]).

Subcellular localisation of [ 177 Lu]Lu-DOTA-TATE
Subcellular localisation of [ 177 Lu]Lu-DOTA-TATE in cells followed the protocol for the uptake assays above, except that, following removal of excess [ 177 Lu]Lu-DOTA-TATE, cells were incubated with ice-cold 0.1 M glycine (pH 2.5) for 10 min prior to pooling with a further PBS wash ("Membrane" fraction).Cells were then detached with Accutase and diluted in medium prior to pelleting and transferring the supernatant to the "Cytoplasm" fraction.The cell pellet was resuspended in ice-cold cell lysis buffer (25 mM KCl, 5 mM MgCl 2 , 10 mM Tris-HCl, and 0.5% v/v IGEPAL CA-630) and incubated for 15 min.Intact nuclei were pelleted and separated from lysed cells by centrifugation (10,000 g, 3 min) and the supernatant removed and combined with the previous portion of the "Cytoplasm" fraction.Pelleted nuclei were resuspended in phosphate-buffered saline (PBS) and transferred to tubes to gamma counting ("Nuclear" fraction).The percentage distribution of [ 177 Lu]Lu-DOTA-TATE in cells was then calculated for the membrane, cytoplasm, and nucleus.

Viability studies after chemotherapeutic agents
U2OS/U2OS + SSTR2A and BON1/BON1 + SSTR2A cells were seeded in 96-well plates at 2 × 10 3 and 4 × 10 3 cells/well, respectively, and allowed to adhere overnight in the incubator.Cells were then treated with a range of hydroxyurea, gemcitabine, and triapine concentrations for 24 h.Hydroxyurea in U2OS/U2OS + SSTR2A cells was used at 152 µg/mL for 24 h [21].Chemotherapeutics were then removed, and cells washed with PBS prior to returning to the incubator in fresh medium.Cells were then grown for a further 5 days post-chemotherapeutics prior to assessing metabolic viability, as described above.Chemotherapeutic concentrations needed to inhibit population growth by 50% (GI 50 ) were then calculated.

Cell cycle, SSTR2A, and size changes post chemotherapeutics
All studies were performed in cells at 4 h after removal of each chemotherapeutics treatment.
For SSTR2A expression studies, 1.5 × 10 5 U2OS/ U2OS + SSTR2A and BON1/BON1 + SSTR2A cells were seeded in 6-well plates and placed in the incubator to adhere overnight.Cells were treated with chemotherapeutics for 24 h at the same concentrations stated above.Determination of SSTR2A expression levels by flow cytometry post chemotherapeutics was performed as described in the Supplementary methods.
For cell size studies, cells were detached by Accutase, washed with PBS and resuspended in medium prior to live-cell analysis by flow cytometry (BD LSRFortessa™).General cell size information was acquired using the forward-scatter laser.

Statistics
Data are presented as mean ± standard deviation.Statistical analysis was carried out using GraphPad Prism v9.1.0and differences were deemed statistically significant at p-values < 0.05.In uptake and viability studies, the Kruskal-Wallis analysis of variance (ANOVA) test was carried out to determine statistical significance.One-way ANO-VAs comparing all chemotherapeutics against untreated in a cell line were used in viability studies.
Combining hydroxyurea, gemcitabine or triapine with [ 177 Lu]Lu-DOTA-TATE in parental U2OS and BON1 cells did not affect viability, highlighting the requirement of internalization of [ 177 Lu]Lu-DOTA-TATE to see potentiation of it through other therapies (Fig. 3).However, potentiation of radiation effects was seen for U2OS and U2OS + SSTR2A cells pre-treated with hydroxyurea, gemcitabine or triapine before X-ray radiation (Figure S7), confirming the radiosensitizing effects of the chemotherapeutics in these cells.
Each chemotherapeutic was also found to increase SSTR2A expression (Fig. 6A-B).For U2OS + SSTR2A cells, mean increases in SSTR2A expression were 46.9 ± 28.2%, 20.8 ± 29.7%, and 36.2 ± 32.4%, while for BON1 + SSTR2A cells mean increases were calculated at 48.8 ± 12.0%, 39.6 ± 45.4% and 119.3 ± 40.9% from baseline expression levels for cells when pre-treated with hydroxyurea, gemcitabine, and triapine, respectively.Chemotherapeutic treatments also increased cell size (Fig. 6C-D).For example, forward-scatter laser median fluorescence intensity for U2OS + SSTR2A cells treated with hydroxyurea, or triapine were 121,835 ± 1,738, 127,616 ± 799 or 126,635 ± 962, respectively, whereas baseline median fluorescence intensity was 101,568 ± 2,081.Cell size did depend on the cell cycle phase (Figure S8), with cells in G1 showing the smallest size and cells in late S-phase (after DNA duplication) showing the largest cell size in BON1 + SSTR2A cells.It is also noted that relative cell sizes of chemotherapeutic pre-treated cells, at all phases of the cell cycle, were consistently larger than cells from baseline cultures.

Discussion
Here, studies were performed with U2OS/ U2OS + SSTR2A and BON1/BON1 + SSTR2A cells, which have previously been useful to study the therapeutic efficacy of [ 177 Lu]Lu-DOTA-TATE.Here, using transfected as well as parental cell lines also provided an opportunity to investigate targeted direct effects from This highlights the specificity of [ 177 Lu]Lu-DOTA-TATE to the SSTR2A receptor as well as the dependence on radioactive 177 Lu and its requirement for this to be cellbound and internalized so as to exert a cytotoxic effect in NET cells.
Pre-treatment of U2OS + SSTR2A cells with hydroxyurea, gemcitabine, or triapine enhanced cellular uptake of [ 177 Lu]Lu-DOTA-TATE, increasing by double or more than observed at baseline levels.Correspondingly, metabolic viability was decreased in cells when [ 177 Lu] Lu-DOTA-TATE was combined with each chemotherapeutic.Our work complements previous studies carried out by Nayak et al. who similarly reported an enhanced uptake of SSTR2A-targeting [ 177 Lu]Lu-DOTA-TOC in cells pre-treated with gemcitabine, which was linked to a reduced cell viability compared to cells treated with [ 177 Lu]Lu-DOTA-TOC alone [22].The mechanism behind the potentiation of [ 177 Lu]Lu-DOTA-TATE by these chemotherapeutics remains unclear though and our work does not determine whether this enhanced treatment efficacy is synergistic or additive.Now that initial concentrations of chemotherapeutics have been established, future combination index studies can be performed to determine whether the effects of chemotherapeutics and [ 177 Lu]Lu-DOTA-TATE are additive or synergistic in nature.
Here, we also found that cytoplasmic localization of [ 177 Lu]Lu-DOTA-TATE remained unaltered.This suggests that there is no significant change in trafficking of [ 177 Lu]Lu-DOTA-TATE upon cellular interaction following chemotherapeutics; therefore it can be determined that subcellular localization was not a significant determining factor in the potentiation observed for [ 177 Lu] Lu-DOTA-TATE cytotoxicity when combined with the chemotherapeutics used.
The ribonucleotide reductase inhibitors, hydroxyurea, gemcitabine, and triapine, have also previously been described as radiosensitisers [12].This effect was observed here in both U2OS and U2OS + SSTR2A cells; indeed, all three chemotherapeutics sensitised cells to X-ray radiation.Whilst it is possible that the chemotherapeutics radiosensitised cells to [ 177 Lu]Lu-DOTA-TATE PRRT by enhancing the likelihood of unrepaired DNA damage as it did for X-rays, here, we showed that they also increased levels of SSTR2A expression.Work using other chemotherapeutics, namely 5-fluorouracil, temozolomide and streptozotocin, also reported increased SSTR2A expression in NET cells and enhanced [ 177 Lu]Lu-DOTA-TATE uptake and cytotoxicity compared to [ 177 Lu]Lu-DOTA-TATE monotherapy [23].It is therefore likely that alongside other factors described below, enhanced SSTR2A expression is a main driver for increased [ 177 Lu]Lu-DOTA-TATE uptake and therefore toxicity.Whether the same would hold true in cancer cells that express SSTR2A naturally, remains to be seen.As SSTR2A expression is naturally seen at lower levels in a variety of healthy tissues also [24,25], it would also be of great interest to determine whether similar enhancements in receptor expression post chemotherapeutics are tumour-specific or happen in healthy tissues too.This could enhance the probability of normal tissue complications and thereby influence maximal administered [ 177 Lu] Lu-DOTA-TATE activities for this therapeutic strategy.By using transfected models, even non-NET cells, this work contributes knowledge towards the feasibility of the metronomic chemotherapeutic/PRRT combination approach in scenarios where SSTR2 expression is high or low.Further work in this area would enable us to determine whether, and importantly, at what point, there may be a cut-off for therapeutic benefit for such a combination approach.
As well as affecting SSTR2A expression, a larger proportion of cells remained in S-phase after having been treated with the chemotherapeutics.This is not uncommon as various therapies have been shown to delay progression through the S-phase.The availability of sister chromatids and consequently homologous recombination repair, particularly during late S-phase contributes to a relative radioresistance [26,27], and therefore it is unlikely that the enhanced cytotoxicity observed in the combination therapy studies here is solely attributable to the cell cycle phase.Another observation in the studies carried out here is an increase in the size of cells treated with the chemotherapeutics.Cells naturally increase in size as they progress through the cell cycle from G 1 to S and G 2 /M.In addition to this natural variation in cell size, chemical inhibition of cellular progression into or through the S-phase with agents such as those employed here are known to introduce a mismatch in metabolic balance, through continued RNA and protein synthesis during this temporary inhibition [15,28], creating the same trend in cell sizes, but at an elevated level at all phases of the cell cycle compared to those observed at baseline.The significance and impact of this on radiosensitisation remains unclear.However, increases in overall cell size, and therefore changes in subcellular compartment size (and [ 177 Lu]Lu-DOTA-TATE localisation within them), could influence the accessibility of SSTR2A receptors and consequently the amount of [ 177 Lu]Lu-DOTA-TATE that binds (separately to enhanced SSTR2A expression).This in turn could affect the amount internalised into cells and/or the biological effectiveness of [ 177 Lu]Lu-DOTA-TATE.As mentioned, the exact influence of cells size remains unclear and to determine the influence of cell size of cytotoxicity from [ 177 Lu]Lu-DOTA-TATE requires further work and cellular dosimetry calculations.
There are very few studies looking into metronomic chemotherapeutic and PRRT combination therapies, although the Lu-X trial shows that adding a chemotherapeutic at metronomic levels with PRRT is a valid approach that minimizes toxicity [29].But it remains difficult to compare our work to clinical trials such as Lu-X where capecitabine was combined with [ 177 Lu]Lu-DOTA-TATE in patient with neuroendocrine tumours [29], as chemotherapy is administered at regular intervals in parallel and surrounding the first/final [ 177 Lu]Lu-DOTA-TATE administrations.However, in our study, we investigated the acute effects of this combination strategy.
Although promising, the in vitro data acquired here needs to be validated in preclinical studies and the optimal regimen needs to be determined.The chemotherapeutic concentrations used here were chosen based on their ability to modulate SSTR2A expression and cell size close to the respective chemotherapeutics' GI 50 values.Further studies should test more concentrations per chemotherapeutic, particularly those lower than used here, and with a further variety of [ 177 Lu]Lu-DOTA-TATE incubation activities.Also, it would be interesting to determine whether other chemotherapeutics have a similar effect on [ 177 Lu]Lu-DOTA-TATE uptake and cytotoxicity in cells, particularly with the proposed combination strategy, and to ascertain through which mechanism that is realised, e.g.cell cycle phase/synchronisation, modulation of SSTR2A receptor expression, or a mixture thereof.Initial clinical studies would also be needed to determine whether (and for how long) existing chemotherapeutic schedules enhance SSTR2A expression and consequently [ 177 Lu]Lu-DOTA-TATE uptake; this could be achieved using either ex vivo tumour samples or through PET imaging using [ 68 Ga]Ga-DOTA-TATE, for example.Next, clinical combination studies to maximize [ 177 Lu]Lu-DOTA-TATE tumour uptake will need to be carried out, albeit it with a view to ensure that toxicities from this approach remain acceptable.It could be envisaged that administered activities of [ 177 Lu]Lu-DOTA-TATE, when used as part of combination regimen, can be lowered without compromising overall tumour uptake when compared to its administration as a monotherapy.Considering the chemotherapeutics investigated here are already in use in the clinic, with triapine also currently in active phase I and II clinical trials, it is forecast that any such approach combining [ 177 Lu]Lu-DOTA-TATE with hydroxyurea, gemcitabine, or triapine has a relatively low barrier to adoption in the clinic and yet could greatly increase outcome for patients with NETs.

Conclusions
Hydroxyurea, gemcitabine and triapine all increased SSTR2A expression and [ 177 Lu]Lu-DOTA-TATE uptake whilst selectively reducing cell metabolic viability in transfected U2OS + SSTR2A and BON1 + SSTR2A cells compared to [ 177 Lu]Lu-DOTA-TATE monotherapy.Further investigations could transform patient care and positively increase outcomes for patients treated with [ 177 Lu] Lu-DOTA-TATE.

Table 2
GI 50 values for hydroxyurea, gemcitabine and triapine in U2OS and BON1 parental and U2OS + SSTR2Aand BON1 + SSTR2A-expressing cell lines.Values used for further studies are in brackets.GI 50 values for hydroxyurea in U2OS + SSTR2A cells were taken from the literature